This invention relates to immunological methods and devices for detecting analytes in biological samples.
Numerous approaches have been developed for detection of a given analyte in a biological sample. Typical of these methods are the so called xe2x80x9cdipstick,xe2x80x9d xe2x80x9clateral flowxe2x80x9d and xe2x80x9cflow-throughxe2x80x9d devices and methods. The dipstick generally uses a plastic strip with a reagent-containing matrix layered thereon. A test sample is applied to the device, and the presence of the analyte is indicated by a visually detectable signal such as a color-forming reaction. The flow-through device generally uses a porous material with a reagent-containing matrix layered thereon or incorporated therein. Test sample is applied to and flows through the porous material, and analyte in the sample reacts with the reagent(s) to produce a detectable signal on the porous material. These devices are generally encased in a plastic housing or casing with calibrations to aid in the detection of the particular analyte.
Lateral flow assays also utilize a porous membrane for performing analyte detection. Instead of drawing the sample through the membrane perpendicularly, however, the sample is permitted to flow laterally from an application zone to a reaction zone on the membrane surface. The capture reagent is present in the reaction zone, and the captured analyte can be detected by a variety of proto-cols, including direct visualization of visible moieties associated with the captured analyte.
For example, Hochstrasser, U.S. Pat. No. 4,059,407, discloses a dipstick device which can be immersed in a biological fluid to semi-quantitate analyte in the fluid. Semi-quantitation of the analyte is accomplished by using a series of reagent-containing pads wherein each pad in the series will produce a detectable color (i.e., a positive result) in the presence of an increasing amount of analyte.
Deutsch et al. describe a quantitative chromatographic test strip device in U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537. The device comprises a material capable of transporting a solution by capillary action, i.e., wicking. Different areas or zones in the strip contain the reagents needed to produce a detectable signal as the analyte is transported to or through such zones. The device is suited for both chemical assays and binding assays which are typified by the binding reaction between an antigen and its complementary antibody.
Many variations on the Deutsch et al. device have also been disclosed. For example, Tom et al., U.S. Pat. No. 4,366,241 discloses a bibulous strip with an immunosorbing zone to which the test sample is applied. Grubb et al., U.S. Pat. No. 4,168,146 describes the use of a porous test strip material to which is covalently bound an antigen-specific antibody. In performance of an assay, the test strip is immersed in a solution suspected of containing an antigen, and capillary migration of the solution up the test strip is allowed to occur. As the antigen moves up the test strip it binds to the immobilized antigen-specific antibody. The presence of antigen is then determined by wetting the strip with a second antigen-specific antibody to which a fluorescent or enzyme label is covalently bound. Quantitative testing can be achieved by measuring the length of the strip that contains bound antigen.
Numerous variations of the above test strip are disclosed in U.S. Pat. No. 4,435,504 which employs a two enzyme indicator system. U.S. Pat. No. 4,594,327 discloses the addition of a binding agent to whole blood samples which causes the red blood cells to aggregate at the area of the strip adjacent to the air/liquid interface. In addition, U.S. Pat. No. 4,757,004 discloses a means for controlling the shape of the fluid front migrating along the test strip. Also of interest are U.S. Pat. Nos. 4,298,688, 4,517,288 and 4,740,468 which describe sheet-like diagnostic devices comprising one or several strips, arranged behind one another, having zones situated one behind another. Each zone is readily accessible from above and below for the addition of reagents. Such devices can quantitatively determine the amount of an analyte.
In addition, European Publication No. 323,605 discloses an assay device using chromatographic material wherein the test sample can travel from one end to the other by capillary action. The chromatographic material contains an immobilized capture reagent capable of binding to the analyte. The application pad receives the test sample and contains a diffusive indicator reagent capable of migrating from the application pad to the chromatographic material. The indicator reagent is capable of binding to the analyte or the capture reagent. The binding of the indicator reagent results in a detectable signal.
Procedures using chromogenic and fluorescent dyes as labels in biological assay procedures are also known. Typical assay protocols call for direct or indirect binding of a dye label to an analyte or analyte analog in a biological sample, where the presence or absence of the dye at a particular stage of the assay can be determined visually and related to the amount of analyte initially present in the sample. A wide variety of specific assay protocols exist.
A number of those assays utilize naturally colored or dyed particles as a label, where the particles are bound to an antibody or other specific binding substance. Suggested particles include dyed latex beads, dye imbibed liposomes, erythrocytes, metal sols, and the like. The colored particle in such complexes can serve as a visible marker, where separation, capture, or aggregation of the particles is mediated through binding of the antibody or other specific binding substance. The amount of label thus segregated in a particular assay step is related to the amount of analyte initially present in the sample.
For example, U.S. Pat. No. 4,943,522 describes a solid phase lateral flow assay using erythrocytes as a label. U.S. Pat. No. 4,863,875 describes compositions comprising at least ten dye molecules or monomers covalently attached to an antibody through an isocyanate group on the dye. U.S. Pat. No. 4,703,017 describes a solid phase assay device which relies on specific binding of a ligand-label conjugate on a solid support, where the label is disclosed as a particle, such as a liposome, or polymer microcapsule. U.S. Pat. No. 4,608,246 describes assays for typing blood which employ erythrocytes as a labeling agent. U.S. Pat. No. 4,452,886 describes the covalent attachment of photon absorbing or emitting polymers to proteins, such as antibodies and antigens. U.S. Pat. No. 4,373,932 describes labeling of a ligand with an aqueous dispersion of a hydrophobic dye or pigment, or a polymer nuclei coated with such a dye or pigment. U.S. Pat. No. 4,313,734 describes methods of detecting sample analytes by the determination of the metallic label content in the sample. U.S. Pat. No. 4,169,138 describes immunoassays which employ visible particles including undyed microorganisms, bound to polymers which may be of microbial origin.
Other lateral flow protocols include U.S. Pat. No. 4,943,522 directed to a lateral flow device which relies on a nonbibulous support to conduct liquids from one portion of the device to another. PCT Publication WO 92/12428, which is related to the above patent, represents an improvement on that method and device wherein nonbibulous lateral flow is used to conduct visible moieties, especially labeled particles, e.g., dyed latex, red blood cells or liposomes capable of reacting with analyte or a competitor thereto into a capture zone for detection, using a bibulous support made nonbibulous by treatment with a blocking agent. The result is a one-step assay which can be conducted in a very short period of time (typically, within 60 seconds), and wherein the readout is usually available instantaneously upon the sample contacting a capture zone.
Other disclosures of lateral flow assays have also appeared. For example, U.S. Pat. No. 4,861,711 describes a lateral flow assay wherein all components needed for the detection of an analyte are embedded in a single sheet. The lateral flow is referred to as chromatographic behavior. This patent discloses the use of enzyme antibody conjugates and substrates, each separately held in absorbent pads. European Patent Application 306,772 describes a lateral flow device which comprises a chromatographic medium wherein the zone for application of sample and the reaction zone with an immobilized reagent capable of binding the analyte or a label-specific binding material are separated. British Application No. 2,204,398 describes a lateral flow device wherein sample applied to the device picks up labeled reagent and permeates into a detection zone. Labels include gold sols and colored particles. U.S. Pat. No. 4,168,146 describes lateral flow through a test strip which determines the presence of an analyte by addition of a suitable color indicator.
European Application No. 276,152 describes a bibulous matrix lateral flow device which operates on two separate planes. U.S. Pat. No. 4,857,453 describes a device wherein reagents are supplied in breakable containers which are integral to the device. European Patent No. 158,746 and U.S. Pat. No. 4,775,636 describe the use of metal sols as visible detecting labels for specific binding assays.
Furthermore, U.S. Pat. No. 4,703,017, describes test strip devices wherein binders for an analyte and/or a visible tracer are applied to defined areas of a solid support by adsorption or covalent coupling. After application of the binder to one or more test areas of the substrate, the residual binding capacity of the test substrate is saturated or blocked by treatment with one or more types of proteins which do not specifically bind the materials to be employed in the assay. The tracer, when bound under assay conditions to the binder or to the analyte bound to the binder, is visible on the support without further treatment. The test strip is contacted and incubated with a sample containing (or suspected of containing) analyte. A sample strip may be provided with a plurality of test areas. U.S. Pat. No. 4,855,240 describes an assay wherein a sample and a tracer as described in U.S. Pat. No. 4,703,017 are applied at disparate locations on a flat lateral flow device. As noted before, the above devices are contained within a plastic housing or casing.
Applicants have determined that it is useful to construct the lateral flow immunochromatographic assay devices of the present invention without plastic casings. Specifically, a porous material is laminated to provide an end product with an adequate mechanical strength, i.e., a particular thickness and strength characteristic for support. In addition, added strength is also achieved by a plastic cover used on top of the porous material. This device provides a simple, convenient, rapid and direct sample application method to the user with a cost effective design. Because no plastic casing is used, this invention also provides a more environmental friendly device. This device can then be used to detect various analytes in a liquid sample.
Taking advantage of the test device of the present method, the device can be utilized with a method for detection of analytes directly from a biological sample, such as urine. In particular, the invention can be used to detect the presence or absence of human chorionic gonadotropin (xe2x80x9chCGxe2x80x9d). Such detection is useful, in determining a positive or negative pregnancy in women.
The first aspect of the present invention features an immunochromatographic assay device for the detection of the presence or absence of an analyte in a liquid sample. The immunochromatographic assay device comprises a separated sample receiving region which is made of a porous material. The porous material conducts lateral flow of the liquid sample. The sample receiving region is in contact with a separate analyte detection region. Lateral flow of the liquid sample will continue from the sample receiving region to the analyte detection region. The analyte detection region contains a porous material which conducts lateral flow of the liquid sample. The analyte detection region contains mobile labeling reagents located at a discrete situs. It also contains an immobile capture reagent at a discrete situs. In addition, it also contains a control reagent at a discrete control situs. The analyte detection region is also in lateral flow contact with the end flow region. The end flow region contains a porous material which conducts lateral flow of the liquid sample. It is capable of absorbing excess liquid sample.
In the above aspect, the one labeling reagent is capable of forming a complex with the analyte. The second labeling reagent is mobile but does not form a complex with either the analyte or the capture reagent. The capture reagent is capable of binding the analyte labeling reagent complex. The control reagent is capable of binding the second labeling reagent. In addition, the porous materials in the above aspect are laminated with one continuous or separate semi-rigid material of at least 0.005 inches thick. The laminate covers the back only and provides adequate mechanical strength to the device, i.e., it provides support and strength characteristics to the porous material and overall device. The front of the porous materials are partially covered with a plastic material so as to leave a portion of the sample receiving region exposed for sample application. The capture situs and the control situs can be seen through the plastic cover.
The term xe2x80x9canalytexe2x80x9d as used herein refers to a compound or composition to be detected or measured in the test sample. The analyte will have at least one epitope that an antibody or an immunological reactive fragment thereof can recognize. Analyte can include any antigenic substances, haptens, antibodies and combinations thereof. The analyte of interest in an assay can be, for example, a protein, a peptide, an amino acid, a nucleic acid, a hormone, a steroid, a vitamin, a pathogenic microorganism for which polyclonal and/or monoclonal antibodies can be produced, a natural or synthetic chemical substance, a contaminant, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, and metabolites of or antibodies to any of the above substances. One preferred example of a hormone suitable for detection is human chorionic gonadotropin (xe2x80x9chCGxe2x80x9d).
The term xe2x80x9csamplexe2x80x9d as used herein refers to any biological sample that could contain an analyte for detection. Preferably the biological sample is in liquid form or can be changed into a liquid form. Preferably, the sample is a urine sample.
As used herein, the term xe2x80x9csample receiving regionxe2x80x9d means the portion of the assay device which is in direct contact with the liquid sample, i.e., it receives the sample to be tested for the analyte in question. The liquid sample can then migrate, through lateral flow, from the sample receiving region towards the end flow region. The sample receiving region is in lateral flow contact with the analyte detection region. This could either be an overlap or end-to-end connection. The analyte in the sample must be capable of migrating, through lateral flow, with the liquid sample. The sample receiving region is made of porous material, usually porous paper.
As used herein, the term xe2x80x9cporous materialxe2x80x9d refers to any material capable of providing lateral flow. This would include material such as nitrocellulose, nitrocellulose blends with polyester or cellulose, untreated paper, porous paper, rayon, glass fiber, acrylonitrile copolymer or nylon. One skilled in the art will be aware of other porous materials that allow lateral flow. The term xe2x80x9clateral flowxe2x80x9d refers to liquid flow in which all of the dissolved of dispersed components of the liquid are carried at substantially equal rates and with relatively unimpaired flow laterally through the material, as opposed to preferential retention of one or more components as would occur, e.g., in materials capable of adsorbing or imbibing one or more components.
The term xe2x80x9cmobilexe2x80x9d as referred to herein means diffusively or non-diffusively attached, or impregnated. The reagents which are mobile are capable of dispersing with the liquid sample and carried by the liquid sample in the lateral flow. The term xe2x80x9cimmobilexe2x80x9d as used herein refers to reagents which are attached to the support such that lateral flow of the liquid sample does not affect the placement of the immobile particle in the discrete region of the porous material. Such attachment can be through covalent or ionic means. Those skilled in the art will be aware of means of attachment to immobilize various particles.
The term xe2x80x9clabeling reagentxe2x80x9d as used herein refers to any particle, protein or molecule which recognizes or binds to the analyte in question or a particle, molecule, protein which does not recognize, or bind to the analyte and has attached conjugated or bound to it, either chemically, covalently or noncovalently, ionicly or nonionicly any substance capable of producing a signal that is detectable by visual or instrumental means. Such labels producing a signal would include chromogens, catalysts, fluorescent compounds, colloidal metallic and nonmetallic particles, dye particles, enzymes or substrates, organic polymers, latex particles, liposomes with signal producing substances and the like. The particle or molecule recognizing the analyte can be either natural or non-natural, preferable monoclonal or polyclonal antibody. One labeling reagent can be a monoclonal antibody to the xcex2-epitope of hCG and bound to Blue latex and another labeling reagent can be BSA bound to red color latex.
The term xe2x80x9ccapture reagentxe2x80x9d as used herein refers to any particle or molecule which recognizes or binds the analyte in question. The capture reagent is capable of forming a binding complex with the labeling reagent that has binded to the analyte in the sample. The capture reagent is immobilized to the porous material of the analyte detection region. The capture reagent is not affected by the lateral flow of the liquid sample due to the immobilization to the porous material. The particle of molecule can be natural, or non-natural, i.e., synthetic. Once the capture reagent binds the analyte-labeling reagent complex it prevents the analyte-labeling reagent from continuing with the lateral flow of the liquid sample.
The term xe2x80x9ccontrol reagentxe2x80x9d as used herein refers to any particle or molecule which is capable of binding the labeling reagent which does not recognize or bind the analyte of question in the sample. For example, the labeling reagent may be BSA conjugated to a red latex. The control reagent would be a particle or molecule which recognizes or binds the BSA conjugated red latex labeling reagent. Preferably, the control reagent would be a monoclonal or polyclonal antibody which recognizes BSA. The control reagent is immobilized to the porous material. Once it binds the labeling reagent it immobilizes the labeling reagent and prevents it from continuing lateral flow with the liquid sample. Just as the capture reagent is immobilized in a discrete situs on the porous material of the analyte detection region, the control reagent is also immobilized in a discrete situs on the porous material of the analyte detection region.
The term xe2x80x9canalyte detection regionxe2x80x9d as used herein refers to the portion of the assay device which is in lateral flow contact with the porous material of the sample receiving region and the end flow region. The contact can be an overlap or end-to-end connection. The analyte in the sample must be capable of migrating through lateral flow with the liquid sample. The analyte detection region is made of a porous material just as the sample receiving region is. Preferably, the analyte detection region is made of nitrocellulose. The sample receiving region, the analyte detection region and the end flow region can be made of different material. The analyte detection region can contain the mobile labeling reagents, the immobile capture reagent and the immobile control reagent. In other embodiments the analyte detection region contains only the immobilized control and capture reagents.
The term xe2x80x9cdiscrete capture situsxe2x80x9d or xe2x80x9cdiscrete control situsxe2x80x9d as used herein refers to a defined area in which either the labeling reagents, the capture reagent or the control reagent are impregnated (for the labeling reagents) or immobilized (for the control or capture reagents) to the porous material. The discrete capture situs of the control or the capture reagents provide a discrete visible line from which to view the results of the test. For example, if the one labeling reagent is analyte bound to anti-analyte conjugated to Blue latex, then a discrete blue line will appear at the discrete capture situs if the capture reagent binds and immobilizes the analyte labeling reagent complex. If the second labeling reagent is BSA conjugated to red latex then a discrete line will form at the discrete control situs if the control reagent immobilized the BSA red latex labeling reagent.
The term xe2x80x9cend flow regionxe2x80x9d as used herein refers to the portion of the assay device which is in lateral flow contact with the analyte detection region. The liquid sample migrates to the end flow region. It is capable of absorbing excess liquid sample. The contact with the analyte detection region can be either by overlap or end-to-end connection. This region is made of porous material, usually porous paper.
The term xe2x80x9csemi-rigidxe2x80x9d as used herein refers to the material used to support the porous material of the device. This can be one continuous piece of laminate or separate pieces. The laminate is preferably vinyl but one skilled in the art will recognize that numerous materials can be used to provide the semi-rigid support. The semi-rigid material must at least be of 0.005 inches thick in order to produce the desired adequate mechanical strength or support for the device to function effectively.
The term xe2x80x9cadequate mechanical strengthxe2x80x9d as used herein refers to a desired support to the assay device so as to function properly. This would include a desired thickness to the backing material, as well as bending characteristics when using weighted standards or bending characteristics when using water flow measurements. The adequate mechanical strength is the support achieved for the entire assembled assay device so as to function properly in the collection and analysis of the analyte in the liquid sample. The minimum adequate mechanical strength is a thickness of 0.005 inches thick with a 10 mm bending measurement using a 10 g weight or 5 mm bending measurement using water flow. Such strength characteristics or measurements provide the minimum standard necessary for proper function of the device. The preferred adequate mechanical strength is 0.02 inches thick, 1 mm bending using the 10 g weight measurement or 5 mm bending using the water flow measurements. Such preferred measurements provided sufficient strength and support to the porous material and assay device that no bending is obtained when the assay is used with a urine stream or water flow.
The term xe2x80x9cplastic material,xe2x80x9d or xe2x80x9cplastic cover,xe2x80x9d or xe2x80x9ccoverxe2x80x9d as used herein refers to any plastic material which can cover the porous material of the device. Preferably, this is mylar, however, those skilled in the art will know of various materials that can be used for such purposes. The cover can be one continuous plastic or separate pieces as shown in the figures. It must allow the discrete control and discrete capture situses to be viewed. Thus, if the cover is clear then the result can be viewed through the clear cover. If the cover is not clear, then a window, gap or hole must be used so the results can be viewed. In addition, the cover must leave a portion of the sample receiving region exposed so the sample can be applied to the receiving region.
Other features and advantages of the invention will be apparent from the following detailed description of the invention in conjunction with the accompanying drawings and from the claims.